Potential drug target validation involves determining whether a DNA, RNA or protein molecule is implicated in a disease process and is therefore a suitable target for development of new therapeutic drugs. Drug discovery, the process by which bioactive compounds are identified and characterized, is a critical step in the development of new treatments for human diseases. The landscape of drug discovery has changed dramatically due to the genomics revolution. DNA and protein sequences are yielding a host of new drug targets and an enormous amount of associated information.
The identification of genes and proteins involved in various disease states or key biological processes, such as inflammation and immune response, is a vital part of the drug design process. Many diseases and disorders could be treated or prevented by decreasing the expression of one or more genes involved in the molecular etiology of the condition if the appropriate molecular target could be identified and appropriate antagonists developed. For example, cancer, in which one or more cellular oncogenes become activated and result in the unchecked progression of cell cycle processes, could be treated by antagonizing appropriate cell cycle control genes. Furthermore many human genetic diseases, such as Huntington's disease, and certain prion conditions, which are influenced by both genetic and epigenetic factors, result from the inappropriate activity of a polypeptide as opposed to the complete loss of its function. Accordingly, antagonizing the aberrant function of such mutant genes would provide a means of treatment. Additionally, infectious diseases such as HIV have been successfully treated with molecular antagonists targeted to specific essential retroviral proteins such as HIV protease or reverse transcriptase. Drug therapy strategies for treating such diseases and disorders have frequently employed molecular antagonists which target the polypeptide product of the disease gene(s). However the discovery of relevant gene or protein targets is often difficult and time consuming.
One area of particular interest is the identification of host genes and proteins that are co-opted by viruses during the viral life cycle. The serious and incurable nature of many viral diseases, coupled with the high rate of mutations found in many viruses, makes the identification of antiviral agents a high priority for the improvement of world health. Genes and proteins involved in a viral life cycle are also appealing as a subject for investigation because such genes and proteins will typically have additional activities in the host cell and may play a role in other non-viral disease states.
Viral maturation involves the proteolytic processing of the Gag proteins and the activity of various host proteins. It is believed that cellular machineries for exo/endocytosis and for ubiquitin conjugation may be involved in the maturation. In particular, the assembly, maturation, budding and subsequent release of retroid viruses, RNA viruses and envelope viruses, such as various retroviruses, rhabdoviruses, lentiviruses, and filoviruses may involve the Gag polyprotein. After its synthesis, Gag is targeted to the plasma membrane where it induces budding of nascent virus particles.
The role of ubiquitin in virus assembly was suggested by Dunigan et al. (1988, Virology 165, 310; Meyers et al. 1991, Virology 180, 602), who observed that mature virus particles were enriched in unconjugated ubiquitin. More recently, it was shown that proteasome inhibitors suppress the release of HIV-1, HIV-2 and virus-like particles derived from SIV and RSV Gag. Also, inhibitors affect Gag processing and maturation into infectious particles (Schubert et al 2000, PNAS 97, 13057; Harty et al. 2000, PNAS 97, 13871; Strack et al. 2000, PNAS 97, 13063; Patnaik et al. 2000, PNAS 97, 13069).
It is well known in the art that ubiquitin-mediated proteolysis is the major pathway for the selective, controlled degradation of intracellular proteins in eukaryotic cells. Ubiquitin modification of a variety of protein targets within the cell appears to be important in a number of basic cellular functions such as regulation of gene expression, regulation of the cell-cycle, modification of cell surface receptors, biogenesis of ribosomes, and DNA repair. One major function of the ubiquitin-mediated system is to control the half-lives of cellular proteins. The half-life of different proteins can range from a few minutes to several days, and can vary considerably depending on the cell-type, nutritional and environmental conditions, as well as the stage of the cell-cycle.
Targeted proteins undergoing selective degradation, presumably through the actions of a ubiquitin-dependent proteosome, are covalently tagged with ubiquitin through the formation of an isopeptide bond between the C-terminal glycyl residue of ubiquitin and a specific lysyl residue in the substrate protein. This process is catalyzed by a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), and in some instances may also require auxiliary substrate recognition proteins (E3s). Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin may be attached to lysine side chains of the previously conjugated moiety to form branched multi-ubiquitin chains.
The conjugation of ubiquitin to protein substrates is a multi-step process. In an initial ATP requiring step, a thioester is formed between the C-terminus of ubiquitin and an internal cysteine residue of an E1 enzyme. Activated ubiquitin may then be transferred to a specific cysteine on one of several E2 enzymes. Finally, these E2 enzymes donate ubiquitin to protein substrates, typically with the assistance of a E3 protein, also known as a ubiquitin ligase enzyme. In certain instances, substrates are recognized directly by the ubiquitin-conjugated E2 enzyme. Ubiquitin (ub) protein ligases (E3's) are functionally defined as proteins that facilitate the covalent linkage (conjugation) of one or multiple ubiquitin molecules to a substrate protein in the presence of E1 (ub-activating enzyme) and an E2 (ub carrier protein). In the absence of a protein substrate, E3's can catalyze self-ubiquitination, that is, transfer of activated ubiquitin from E2 to a lysine residue acceptor site on the E3 polypeptide, a reaction termed self-ubiquitination. Similar to trans ubiquitination, self-ubiquitination is dependent on the presence of E1, E2 and an intact E3 functional module i.e. RING finger or HECT domain (Lorick K L et al., Proc Natl Acad Sci USA. 1999 96:11364-9; Kao W H et al., J. Virol. 2000 74:6408-6417).
It is also known that the ubiquitin system plays a role in a wide range of cellular processes including intracellular transport, cell cycle progression, apoptosis, and turnover of many membrane receptors. In viral infections, the ubiquitin system is involved not only with assembly, budding and release, but also with repression of host proteins such as p53, which may lead to a viral-induced neoplasm. The HIV Vpu protein interacts with an E3 protein that regulates IκB degradation, and is thought to promote apoptosis of infected cells by indirectly inhibiting NF-κB activity (Bour et al. (2001) J Exp Med 194:1299-311; U.S. Pat. No. 5,932,425). The ubiquitin system regulates protein function by both monoubiquitination and polyubiquitination. Polyubiquitination is primarily associated with protein degradation.
POSH (Plenty of SH3 domains) proteins play a role in a wide range of cellular processes including protein degradation, intracellular transport, cell cycle progression, apoptosis, and turnover of many membrane receptors. The essential function of POSH, a ubiquitin ligase, and “POSH proteins” (proteins that inherently include in their amino acid sequence a RING domain and at least one SH3 domain) in viral infection and the use of POSH inhibition to inhibit viral infections and, in particular, HIV infection, were broadly described in U.S. application Ser. No. 10/293,965, filed Nov. 12, 2002; PCT/US02/36366, filed Nov. 12, 2002, published as WO 03/095972; PCT/US02/24589, filed Jul. 31, 2002; WO 03/078601, WO 03/060067, EP 1310552, and EP 02257796, filed Nov. 11, 2002. All these applications are hereby incorporated by reference herein in their entirety as if fully disclosed herein.
A ubiquitin ligase, such as POSH, may participate in biological processes including, for example, one or more of the various stages of a viral lifecycle, such as viral entry into a cell, production of viral proteins, assembly of viral proteins and release of viral particles from the cell. In the patent applications mentioned hereinabove, it has been described that certain POSH polypeptides are involved in viral maturation, including the production, post-translational processing, assembly and/or release of proteins in a viral particle. Accordingly, viral infections may be ameliorated by inhibiting an activity (e.g. ubiquitin ligase activity or target protein interaction) of POSH.
In addition, as described in the application PCT/US2004/10582, filed on Apr. 5, 2004, herein incorporated by reference in its entirety, several proteins interact with POSH and may be used to identify candidate therapeutics. One of these POSH-associated proteins (POSH-APs) is HERPUD1, known to be associated with neurological disorders, and in particular with Alzheimer's disease.
It would be beneficial to identify compounds as small molecules that bind POSH proteins and inhibit POSH protein activity and, more specifically, compounds that inhibit POSH protein-mediated ubiquitination.
Throughout this specification, various scientific publications and patents or published patent applications are referenced. The disclosure of all these publications in their entireties is hereby incorporated by reference into this specification in order to more fully describe the state of the art to which this invention pertains. Citation or identification of any reference in this section or any other part of this application shall not be construed as an admission that such reference is available as prior art to the invention.